Identifying primary and secondary MLH1 epimutation carriers displaying low-level constitutional MLH1 methylation using droplet digital PCR and genome-wide DNA methylation profiling of colorectal cancers

Table 2 The characteristics of the six CRCs in the diagnostically challenging group comprised of carriers of MLH1 promoter VUS (n = 3) and MLH1 methylated early-onset CRCs (n = 3)

Inclusion Group	Dx age (yrs)	Sex	CRC anatomical site	MMR IHC	MSI^e	Mean methylation in the VM-CpGs^d	CIMP in CRC (Total positive)^b	Mean MLH1 promoter methylation (Tumour) ^a	Blood MLH1 promoter methylation (MethyLight)^c	Non-tumour MLH1 promoter methylation (ddPCR)^d	Additional tissue tested (MethyLight)^c	MMR genes LOH/somatic mutation	APC promoter methylation^f	KRAS somatic mutation
MLH1 VUS (MLH1: c.-11C > T)	68	F	Proximal	MLH1/PMS2 loss	MSI-H	0.288	Negative (0/5)	0.43	0%	Blood—0% NMadj—2.3% NMdist—1.2% Buccal—6.5%	–	MLH1 LOH^g	0.12	None detected
MLH1 VUS (MLH1: c.-11C > T)	60	M	Proximal	MLH1/PMS2 loss	MSI-H	0.392	Negative (2/5)	0.39	1%	Blood—4.5% NMadj—3.4% NMdist—nt Buccal—13.9%	Duodenal cancer—29%	MLH1:p.S131Ter	0.46	p.G12D
MLH1 VUS MLH1: c-[28A > G; 7C > T]	35	F	Proximal	MMRp (Incomplete loss of MLH1/PMS2)	MSS	0.282	Negative (1/5)	0.14	0%	Blood—0% NMadj—0% NMdist—0% Buccal—0%	–	None detected	0.14	p.G12A
MLH1me EOCRC	37	F	Proximal	MLH1/PMS2 loss	MSI-H	0.374	Negative (2/5)	0.34	0%	Blood—0% NMadj—nt NMdist—0% Buccal—nt	–	MLH1: p.S108AfsTer28	0.59	p.G12D
MLH1me EOCRC	43	M	Proximal	MLH1/PMS2 loss	MSI-H	0.338	Negative (0/5)	0.42	1%	Blood—< 1% NMadj—nt NMdist—4.1% Buccal—2.4%	TVA—49%	MLH1: p.X701_splice	0.57	None detected
MLH1me EOCRC	31	M	Proximal	MLH1/PMS2 loss	MSI-H	0.353	Negative (1/5)	0.67	0%	Blood—0% NMadj—nt NMdist—nt Buccal—0%	–	MLH1 LOH^g	0.35	None detected

NM_dist denotes distal normal mucosal samples derived from resection margin (i.e. non-adjacent), NM_adj denotes normal mucosal samples derived from histologically normal mucosal samples adjacent to the tumour, TVA Tubulovillous Adenoma
^aThe values indicate mean β-values estimated from 4 CpGs overlapping MLH1 promoter C region [39] derived from the HMEPIC data
^bAs assessed from the HMEPIC
^cAs assessed by MethyLight technique
^dMean methylation levels across the 77,113 most variably methylated CpGs, which was used for defining the Consensus clusters
^eEstimated using MANTIS
^fMean methylation from 16 CpGs across the APC promoter c(hr3:112,072,926–11,207,395)
^gLOH—loss of heterogygosity (across the MLH1 locus). The LOH plot is shown in Additional file 9: Fig. S8

ISSN: 1868-7083